Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBiT/BRET Assessments,ACS Pharmacology & Translational Science

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Quantitative Profiling of WNT-3A Binding to All Human Frizzled Paralogues in HEK293 Cells by NanoBiT/BRET Assessments
ACS Pharmacology & Translational Science ( IF 4.9 ) Pub Date : 2021-05-11 , DOI: 10.1021/acsptsci.1c00084
Paweł Kozielewicz ₁ , Rawan Shekhani ₁ , Stefanie Moser ₂ , Carl-Fredrik Bowin ₁ , Janine Wesslowski ₂ , Gary Davidson ₂ , Gunnar Schulte ₁

Affiliation

The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors—the ten mammalian Frizzleds (FZDs)—remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.

中文翻译：

通过 NanoBiT/BRET 评估定量分析 WNT-3A 与 HEK293 细胞中所有人类卷曲旁系同源物的结合

WNT 信号系统控制胚胎发育和组织稳态期间的关键过程，其功能障碍可导致癌症。关于 19 种哺乳动物 WNT 与其受体的富含半胱氨酸结构域 (CRD) - 10 种哺乳动物 Frizzleds (FZD) 的相对结合亲和力的选择性和差异的细节仍不清楚。在这里，我们使用 eGFP 标记的小鼠 WNT-3A 系统分析 WNT 与 HEK293A 细胞中每个人类 FZD 旁系同源物的相互作用。使用 HiBiT 标记的全长 FZD，我们使用基于 NanoBiT/BRET 的测定法研究了 eGFP-WNT-3A 结合动力学、饱和结合和与市售 WNT 在活 HEK293A 细胞中的竞争结合。此外，我们生成了受体嵌合体来剖析跨膜核心对 WNT-CRD 结合的贡献。我们的数据确定了不同的 WNT-FZD 选择性，并阐明了复杂的 WNT-FZD 结合机制。本文描述的方法学发展揭示了 WNT 信号传导和 WNT-FZD 相互作用的复杂性尚未得到认可的细节，提供了有关 WNT-FZD 选择性的更多细节。

更新日期：2021-06-11

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